User:Saroj Pandey/Notebook/SNP PCR optimization/2014/11/13

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Allele Specific PCR

Four different forward primers were used in combination with the two specific reverse primers for taster and non-taster.

saliva PCR

electrophoresis

Forward primers used:

EP9_F: AGCTATGCCCCCTTTCCTCT

EP7_F: TGGAAAGGAACCATGCATTT

GaF : ATCCGTGATGCTGTGCTATG

EP3_F: CCCTCTAAGTTTCCTGCCAGA

Reverse primers used:

Cfb(R): CAATCACTGTTGCTCAGTGG

Gfb(R): CAATCACTGTTGCTCAGTGC

Expected product sizes:

911bp, 760bp, 510bp, 356bp

Observation

• For taster, first three combinations of the primers gave bands at specific positions but the EP_3+Cfb(R) did not give specific band. This is because the enzyme was not added to the mixture during the PCR. So the amount of unspecific product is much higher.

• All the primer combinations worked and showed specific bands at correct positions for non-taster.

• Unspecific primers did not produce bands as with the specific ones. So the separation was successful.

Conclusion

• With these primer combinations, the taster and non-taster of the phenylthiocarbamide (PTC) can be identified genotypically.